Reactive oxygen intermediates (ROI), which include the superoxide anion (O2−) and hydrogen peroxide (H2O2), result from the stepwise, monovalent reduction of oxygen: O2− results from the addition of a single electron to O2, and H2O2 results from the addition of a single electron to O2−. ROI are cytotoxic and mutagenic, with high levels of ROI causing damage to biomolecules such as DNA, proteins, and biomembranes. However, recent data indicate that lower levels of ROI may function in signal transduction as intracellular mediators of cell growth, cell proliferation, angiogenesis, apoptosis, and senescence.
Several biological systems generate ROI. For example, within the phagocytic-based immune defense against invading microbes, cells such as neutrophils produce large quantities of ROI via the nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) oxidase (also known as the respiratory burst oxidase). The catalytic subunit of this enzyme, gp91phox, oxidizes NADPH and reduces oxygen to form O2−.
In many non-phagocytic cell types including cells in the colon, lung, brain and kidney, the gp91phox homologue family of NADPH oxidase (NOX) and dual oxidase (Duox) enzymes is responsible for producing low levels of ROI. At present, six human homologues of gp91phox have been identified, with additional homologs present in rat, mouse, Caenorhabditis elegans, and Drosophila. 
Although the functions of NOX and Duox-derived ROI are unclear, several studies suggest that the non-phagocytic generation of ROI modulates cellular proliferation and activation of growth-related signaling pathways. For example, both fibroblasts and endothelial cells produce increased levels of superoxide in response to cytokines such as interleukin-1 and tumor necrosis factor (TNF). In rat vascular smooth muscle cells, exposure to platelet-derived growth factor (PDGF) increases the release of H2O2 while concomitantly increasing cell proliferation (Meier et al., Biochem. J. 263:539-45 (1989); Matsubara et al., J. Immun., 137:3295-98 (1986)). Additionally, data show that low levels of ROI elicit downstream effects on the redox-sensitive transcription factor nuclear factor kappa-B (NFκ-B) and activator protein-1 (AP-I) (Schreck et al., EMBO J., 10:2247-58 (1991); Schmidt et al., Chemistry & Biology, 2:13-22(1995)).
Non-phagocytic ROI appear to have a direct role in regulating cell division, and may function as mitogenic signals in pathologic conditions related to cell growth, such as cancer and cardiovascular disease. For example, cytokine-mediated endothelial production of O2− may play a role in angiogenesis (Matasubara et al., J. Immun., 137:3295-98 (1986)). Matasubara et al. have proposed that O2− and H2O2 function as “life signals”, preventing cells from undergoing apoptosis. (Matasubara et al., J. Immun., 137:3295-98 (1986)). Other data suggest that ROI mediate both pro-apoptotic and pro-survival signals. (Garg & Aggarwal, Mol. Immunol., 39:509-17 (2002)).
NOX Enzymes
A series of overexpression studies using the NOX1 enzyme of the NOX family of proteins indicates a specific role for NOX1-derived ROI in pathological conditions related to cell growth and proliferation. For example, overexpression of NOX1 in fibroblasts induces an H2O2-dependant malignant transformation, resulting in a highly tumorigenic phenotype (Arnold et al., Proc. Natl. Acad. Sci. USA, 89:5550-55 (2001); Suh et al., Nature (London), 401:79-82 (1999)). Consistent with these findings, overexpression of NOXI in prostate epithelial cells has been found to increase tumorigenicity. This increased epithelial cell tumorigenicity is also associated with increased tumor vascularity and increased expression of vascular endothelial growth factor (VEGF), indicating a specific role for NOX1 in angiogenesis. (Arbiser et al, Proc. Natl. Acad. Sci. USA, 99:715-20 (2002)).
One specific pathophysiological condition that may involve ROI is colorectal cancer (CRC), a form of cancer highly prevalent in the Western world. Sporadic colorectal cancer, which accounts for approximately 85% of diagnosed CRC, is linked to somatic mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Genetic analyses of adenoma-carcinoma sequences for CRC indicate that mutations in APC are common, and may be the triggering event for the disease. The identification of a role for APC in CRC arises from the discovery of germline mutations in APC that result in the rare inherited form of colorectal cancer, familial adenomatous polyposis (FAP). Even though APC mutations may serve as a trigger for CRC, the fact that families carrying identical mutations in APC often exhibit varying degrees of colorectal cancer, both in severity and onset, suggests that other factors influence the function of APC. In other words, although a mutational loss of APC function may predispose an individual to colon cancer, other factors, such as ROI, may ultimately determine the onset and severity of CRC.
Duox Enzymes
Dual oxidases, or Duox, have both a peroxidase-homology domain and a gp91phox domain. It is currently believed that Duox enzymes have dual enzymatic functions, catalyzing both the generation of superoxide and peroxidative type reactions. The latter class of reactions utilizes hydrogen peroxide as a substrate. Since hydrogen peroxide is generated spontaneously from the dismutation of superoxide, it is believed that the NAD(P)H oxidase domain generates the superoxide and/or hydrogen peroxide which can then be used as a substrate for the peroxidase domain. The peroxidase domain is likely to confer additional biological functions. Depending upon the co-substrate, peroxidases can participate in a variety of reactions including halogenation such as the generation of hypochlorous acid (HOCl) by myeloperoxidase and the iodination of tyrosine to form thyroxin by thyroid peroxidase. Peroxidases have also been documented to participate in the metabolism of polyunsaturated fatty acids, and in the chemical modification of tyrosine in collagen. Duox is also theorized to function in the formation or modification of extracellular matrix or basement membrane. Since the extracellular matrix plays an important role in tumor cell growth, invasion and metastasis, it is believed that the Duox type enzymes play a pathogenic role in such conditions.
Although a strong link exists between NOX and Duox enzymes and ROI function in a multitude of different physiological and pathophysiological conditions, in vivo models to study this link in a tissue-specific fashion are lacking. Without such models, the extent to which NOX or Duox-generated ROI participate in cellular proliferation and activation of growth-related signaling pathways in different tissue types is difficult to ascertain. Similarly, the identification of NOX or Duox enzyme regulatory molecules is difficult in the absence of such models.
Accordingly, what is needed are in vivo and in vitro models to examine the effect of NOX and Duox-derived ROI on cell proliferation and activation of growth-related signaling pathways. Additionally, what is needed are in vivo and in vitro models to examine the effect of NOX and Duox-derived ROI on cell proliferation and activation of growth-related signaling pathways in a tissue specific manner. Also needed are in vivo and in vitro models to identify the regulators of NOX and Duox activity, including models to test the ability of different compounds to modulate the function of NOX and Duox and the effect of NOX and Duox-derived ROI on cellular proliferation and activation of growth-related signaling pathways.